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Leukocyte immunoglobulin-like receptors (LILRs) on neutrophils

Periodic Reporting for period 1 - NEUTRO-LILR (Leukocyte immunoglobulin-like receptors (LILRs) on neutrophils)

Reporting period: 2016-10-03 to 2018-10-02

The NEUTRO-LILR project is a Marie-Curie actions Individual Intra-European Fellowship (IEF) based at the Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands. Neutrophils provide the first line of immune defense against invading microbes, and have key roles in orchestrating inflammation. Balanced neutrophils responses are critical to providing immunity and to prevent host tissue damage. A better understanding of the mechanisms that modulate neutrophils is required to identify the best strategies for modulation their activity and functions in disease situations. The project explored the role of the leukocyte immunoglobulin-like receptor (LILR) family in regulating neutrophils. Improved understanding of the role of LILRs as modulators of neutrophil functions, and the identification of novel LILR ligands, is vital to recognizing mechanisms and identifying tools that can be exploited in future immunotherapeutic strategies. This is important for identify the ebst strategies for immunomodulation of immune responses in infections or inflammatory disease situations. Our research demonstartes that LILRs modulate both activation and effector functions of neutrophils, and we have identified new LILR agonists and antagonists that may serve as the basis of new LILR targeting therapeutics.
Project Aims
The Project Aims, as described in Part B of the grant agreement, are as follows:-
1) to understand how LILR stimulation modulates neutrophil functions
2) to identify bacterial molecules which bind LILRs
3) assess whether neutrophil functions can be modulated with LILR ligands

Progress on Project Aims
In work package (WP)1, we aimed to use a series of assays to determine how activation of LILR on neutrophils modified their activation and effector functions. We commenced WP1 by characterizing the expression of LILR on the surface of neutrophils using LILR-specific mAb, and identified that 7 of 11 members of the LILR family could be readily and repeatedly detected at the cell surface. As planned, we completed characterization of the ability of LILR to modulate neutrophil activation, phagocytosis and degranulation. The major finding was that LILR activation is able to modulate Fc receptor mediated activation and effector functions of neutrophils. This work will soon be submitted for publication, and has generated new international collaborations for the fellow.
In WP2, we aimed to identify bacterial molecules which bind LILR. In task 1, we planned to take two independent approaches – first, to assess the ability of bacteria to directly interact with LILR, and a second, more high-risk, phage display secretome library screen to identify bacterial molecules that interact with LILR. During the initial phases of task 1, it became clear that Group B Streptococcus (GBS) interacted with LILR. We focused our time and resources in characterizing the molecular basis of this interaction, and abandoned our plans to screen phage display seceretome libraries as this was a highly time- and cost- consuming screen. We successfully identified the GBS protein responsible for forming the LILR interaction, and successfully expressed recombinant forms of these ligands in task 2. Multiple international collaborations have been formed in this research, and we are currently exploring various aspects of this work. We envisage that the research will be submitted for publication within 6-12 months of the NEUTRO-LILR project finishing. These novel ligands provide new resources to study LILR functions, as well as providing new knowledge of GBS host-pathogen interactions and virulence strategies. In task 3, we performed bioinformatics analysis to identify if homologs of the LILR ligands exist in other bacterial species. Our data suggests that homologs of our identified ligand are not in non-Streptococcal bacteria.
In WP3, we aimed to assess whether neutrophil functions can be modulated with novel LILR ligands. We have generated LILR-reporter cell lines and characterized the agonistic and antagonistic properties of our newly identified LILR ligands. These ligands now provide new tools for studying LILRs in immune cell biology. Finally, we tested whether these ligands could modulate neutrophil activation and effector functions through LILR-specific pathways. This work is currently on going, and our preliminary experiments suggest that these ligands do possess immunomodulatory properties for neutrophils.

Results overview
The NEUTRO-LILR project has improved knowledge of LILRs in neutrophil biology, and identified novel interactions between bacteria and LILR.

Exploitation & Dissemination
Our results will be soon be submitted for publication. At least one manuscript will be submitted from the results of WP1. In addition, it is likely that at least 2 to 3 manuscripts will be submitted for publication based on the results of WP2 and WP3.
Conclusions & Impact
The NEUTRO-LILR project has improved knowledge of LILRs in neutrophil biology, and identified novel interactions between bacteria and LILR. Collectively, the project has provided new resources to study LILR in the future, and improved our understanding of bacterial pathogenesis strategies. It is anticipated that publications resulting from the project will have a great impact on the research community. Data presented at conferences has already attracted attention amongst innate immunity and bacteriology fields, with new collaborations being formed for the future.

Impact of Marie Curie Fellowship on Dr McCarthy’s Career
The Marie Curie fellowship has had many benefits on Dr McCarthy’s career. In 2019, he will commence a faculty position at the MRC Centre for Molecular Bacteriology and Infection, Imperial College London, UK where he will lead is own independent laboratory investigating the roles of LILR in modulating innate immunity. He will continue to collaborate with colleagues at UMC Utrecht, The Netherlands and the international collaborations he has formed during the IEF (colleagues in USA, Germany, Sweden), in order to improve understanding of how the innate immune response is modulated in health and disease.